Glutathione depletion is the earliest detectablebiochemicalevent in the Parkinsonian substantia nigra (SN), occurring prior to selective loss of mitochondrial complex I (CI)activity associated with the disease. We have previously demonstrated that down-regulation of total glutathione (GSH + GSSG) levels in cultured dopaminergic cell lines results in decreased mitochondrial function linked to a selective decrease in CI activity (Jha et al.,2000). Loss of CI activity following acute glutathione depletion appears to be due to reversible nitrosylation of protein subunits comprisingthis complex.The effects of prolonged chronic glutathione depletion on CI activity in dopaminergic cells, however, are unknown but may involve additional, irreversible oxidative events. A major goal of our project is to assess CI inhibition in our dopaminergic cell model at various times and levels of glutathione depletion in order to identify the limitsof function vs. dysfunction including the thresholds for reversible vs. irreversible inhibition as well as the oxidant species and protein targets involved. Once such targets have been identified, we will assess the presence of similar alterations in a newly constructed dox-inducible antiGSH transgenic mouse model following titration of glutathione levels within SN dopaminergic neurons in vivo as a model for molecular events associated with Parkinson's disease. Acute reduction in glutathione in our cell model also results in inhibition of the GSSG reducing enzyme glutathione reductase (GluRd) and up-regulation of the enzyme g-glutamyl transpeptidase (GGT) which breaks down extracellular GSHto substrates which are transported back into the cell for glutathione synthesis. The former may contribute to the detrimental effects of glutathione depletion on mitochondrial function while the latter appears to be a compensatory event. We will also explore the molecular mechanisms involved in these molecular alterations following titration of glutathione pools including the oxidants and the enzyme targets involved and if/how these changes modify mitochondrial function. We will assess the effects of both transgenic GSSG Rd and GGTexpression on CI activity and mitochondrial function in our dopaminergic cell model to see if they act to attenuate any detrimental mitochondrial effects.